- AMER ASSOC CANCER RESEARCH
The activation of protein kinase G (PKG) by cGMP has become considerable interest as a novel molecular mechanism for the induction apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases at thereby increase cellular levels of cGMP appear to induce apoptosis v this mechanism. However, other effects of these compounds have not be excluded, and the precise mechanism by which PKG activation induces apoptosis has not been elucidated in detail. To directly examine the effects of PKG on cell growth and apoptosis, we generated a series of mutants PKG Ialpha: PKG IalphaS65D, a constitutively activated point mutant; PKG Ia a constitutively activated N-terminal truncated mutant; and PKG IalphaK390R, a dominant-negative point mutant. A similar series of mutan of PKG Ibeta were also constructed (Deguchi et aL, Mol. Cancer Ther., 1: 803-809, 2002). The present study demonstrates that when transiently expressed in SW480 colon cancer, the constitutively activated mutants PKG Ibeta, and to a lesser extent PKG la, inhibit colony formation an induce apoptosis. We were not able to obtain derivatives of SW480 cells that stably expressed these constitutively activated mutants, presumably because of toxicity. However, derivatives that stably overexpressed wild- type PKG Ibeta displayed growth inhibition, whereas derivatives that stab expressed the dominant-negative mutant (KR) of PKG Ibeta grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG Ibeta was associate with decreased cellular levels of beta-catenin and cyclin D1 and increased levels of p21(CIP1). Reporter assays indicated that activation of PKG I inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants PKG Ibeta inhibited cell migration. Taken together, these results indicated that activation of PKG Ibeta is sufficient to inhibit growth and cell migration and induce apoptosis in human colon cancer cells and that these effects are associated with inhibition of the transcription of cyclin D1 and an increase in the expression of p21(CIP1).
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